This invention relates to modulators of soft tissue calcification. In particular, this invention relates to the discovery that inhibition of bone resorption will also result in the inhibition of calcification of soft tissues.
The bisphosphonates have been known to chemists since the middle of the 19th century, when the first synthesis occurred in 1865 in Germany (Menschutkin (1865) Ann. Chem. Pharm., 133: 317-320). Bisphosphonates were used in industry, mainly as corrosion inhibitors or as complexing agents in the textile, fertilizer and oil industries. Their ability to inhibit calcium carbonate precipitation, similar to polyphosphates was put to use in the prevention of scaling (Blomen (1995) Pages 111-124 in Bijvoet OLM et al., eds. Bisphosphonate on Bones, Elsevier, Amsterdam).
More recently, bisphosphonates have been developed as drugs for use in various diseases of bone, tooth, and calcium metabolism. The bisphosphonates have two fundamental previously known biological effects: inhibition of calcification when given at high doses and inhibition of bone resorption.
Bisphosphonates have been shown to efficiently inhibit ectopic calcification in vivo. Thus, among others, they prevent experimentally induced calcification of many soft tissues when given both parentally and orally (Fleisch et al. (1970) Eur. J. Clin. Invest., 1: 12-18; Rosenblum et al. (1977) Calcif. Tissue Res., 23: 151-159). In contrast to pyrophosphate, which acts only when given parenterally, bisphosphonates are active when administered orally. They have also been shown to have activity when released locally from various matrices (Levy wet al. (1985) Science, 228: 190-192; Golomb et al. (1986) J. Contr. Rel., 4: 181-194). In addition, topical administration can lead to a decreased formation of dental calculus (Briner et al. (1971) Int. Dent. J. 21: 61-73). This effect is used to prevent tartar formation in humans by the addition of bisphosphonates to toothpastes. In addition, certain bisphosphonates inhibit ectopic ossification when given systemically (Plasmans et al. (1978) Clin. Orthop., 132: 233-243) or locally (Ahrengart and Lindgren (1986) J. Orthop., Res. 4: 18-26).
Of the bisphosphonates, etidronate has been used in humans to prevent ectopic calcification and ossification. Unfortunately with respect to calcification, the results have been disappointing. In conditions such as scleroderma, dermatomyositis, and calcinosis universalis, the results have proven at best inconclusive (Fleisch (1988) Pages 440-466 in Baker PF (ed) Handbook of Experimental Pharmacology, Springer-Verlag, N.Y.). In urolithiasis, the dose that was believed to potentially be effective was such that normal bone mineralization was inhibited (Baumann et al. (1978) Clin. Sci. Mol. Med., 54: 509-516). Other reports also describe the effects of bisphosphonates on ectopic ossification, especially fibrodysplasia ossificans progressiva (Reiner et al. (1980) Pages 237-241 in Caniggia A (ed) Etidronate. Instituto Gentili, Pisa.), and ossification after spinal cord injury, cranial trauma, and total hip replacement (Slooff et al. (1974) Acta Orthop. Belg. 40: 820-828; Finerman and Stover (1981) Metab. Bone Dis. Relat. Res., 4: 337-342; Thomas and Amstutz (1985) J. Bone Joint Surg. (Am) 67: 400-403). While such studies have raised the hope that bisphosphonates might be used clinically to inhibit various types of calcifications, when administered in doses approximating those that inhibit soft tissue calcification, bisphosphonates have impaired the mineralization of normal calcified tissues such as bone and cartilage (King et al. (1971) Clin. Orthop., 78: 251-270; Schenk et al. (1973) Calcif. Tissue Res., 11: 196-214; Flora et al. (1980) Metab. Bone Dis. Rel. Res., 2: 389-407), and, when given in higher amounts, also dentine (Larsson (1974) Calcif. Tiss. Res., 16: 109-127), enamel (Ogawa (1980) Jpn. J. Oral Biol., 22: 199-226; Weile et al. (1990) Arch. Oral Biol., 22: 199-226), and cementum (Alatli and Hammarstrom (1996) Acta Odontol. Scand., 54: 59-65).
Moreover, while the different bisphosphonates vary greatly in their activity in bone resorption, they do not vary greatly in the inhibition of mineralization. For most bisphosphonates, the effective daily dose was believed to be on the order of 5-20 mg of compound phosphorus per kg, administered parenterally, suggesting that the bisphosphonates inhibit calcification at high doses via a common mechanism.
Thus, although bisphosphonates have proven successful when administered to humans or other mammals to inhibit bone resorption, the propensity to inhibit the calcification of normal bone when administered at dosages believed high enough to inhibit ectopic calcification, has hampered the therapeutic use of bisphosphonates in the treatment of ectopic calcifications.
This invention provides new approaches to the treatment of ectopic calcifications and various arterioscleroses (e.g., atherosclerosis). The methods of this invention are premised, in part, on the discovery that agents that inhibit bone resorption will also inhibit ectopic calcification and/or plaque formation and related pathologies associated with arteriosclerosis. Without being bound to a particular theory, it is believed that the process of bone resorption, delivers solubilized calcium (e.g. in a calcium phosphate/protein complex) to the blood where it can travel to sites far removed from bone and there act as a nucleation complex for the formation of ectopic calcifications or atherosclerotic plaques and/or contribute to the formation of an existing calcium deposition.
Various agents, in particular bisphosphonates, are often able to inhibit bone resorption at far lower dosages than the dosages at which they have been observed to inhibit bone calcification. It was believed that the effect on bone resorption was mediated via a biological/cellular mechanism and the effect on bone calcification was mediated by a physio-chemical mechanism (e.g. direct binding to hydroxyapatite). Similarly, it was believed that bisphosphonates could inhibit ectopic calcification by the same physio-chemical mechanism as that used to inhibit bone mineralization. Consequently it was believed that although high dosages of bisphosphonates could inhibit ectopic calcification, this approach had little therapeutic value because of the adverse effect on bone mineralization.
The discovery of this invention, that ectopic calcification can be inhibited by inhibition of bone resorption allows the treatment of pathologies associated with undesired calcification at low dosages, e.g. at dosages that do not adversely effect bone mineralization. Thus, in view of the discoveries described herein, a new therapeutic modality is provided for the alleviation of ectopic calcifications and/or arteriosclerotic plaque formation.
Thus, in one embodiment, this invention provides methods of inhibiting calcification of a soft tissue (e.g., an artery, a heart valve, an atherosclerotic plaque, a cancer, a kidney, a prostate, skin, muscle, cartilage, viscera, and heart muscle) in a mammal. These methods involve inhibiting osteoclastic bone resorption in said mammal (e.g., a mammal diagnosed as having or at risk for a pathology characterized by calcification of a soft tissue) The inhibition is preferably by administration of a bisphosphonate to the mammal in a concentration sufficient to inhibit bone resorption without inhibiting bone mineralization. In preferred embodiments, the bisphosphonate effects a significant reduction of bone resorption at a concentration at least 10-fold, more preferably at least 100-fold, and most preferably at least 1000-fold lower than the concentration at which said bisphosphonate effects a significant reduction of bone mineralization (preferably in the same assay and at the same confidence level). The bisphosphonate may be administered at a dosage at least 10-fold, more preferably at least 100-fold, and most preferably at least 1000-fold lower than concentration at which said bisphosphonate effects a significant reduction of bone mineralization (preferably in the same assay and at the same confidence level). Particularly preferred bisphosphonates include, but are not limited to alendronate, ibandronate, zoledronate, incadronate, risedronate, EB-1053, neridronate, olpadronate, pamidronate, YH 529, tiludronate, and clodronate.
In another embodiment this invention provides methods of method of inhibiting calcification of soft tissue (e.g., an artery, a heart valve, an atherosclerotic plaque, a cancer, a kidney, a prostate, skin, muscle, cartilage, viscera, and heart muscle) in a mammal diagnosed as having or at risk for a pathology characterized by calcification of a soft tissue. These methods involve administering to the animal a low dosage of a bisphosphonate, where the low dosage is sufficient to inhibit the calcification, but below the dosage of the bisphosphonate that inhibits normal bone mineralization. Preferred bisphosphonates and dosages include those described above. In one embodiment the bisphosphonate is alendronate administered at a dosage ranging from the minimum dose that produces a detectable inhibition of bone resorption up to 0.5 mg P/kg/day. In another embodiment, the bisphosphonate is alendronate administered to humans at an oral dosage ranging from 5 mg to 40 mg per day. In still another embodiment, the bisphosphonate is ibandronate administered at a dosage ranging from the minimum dose that produces a detectable inhibition of bone resorption up to 0.5 mg P/kg/day, preferably at an intra venous dosage of 1 mg per day. In still yet another embodiment, the bisphosphonate is zoledronate, incadronate, risedronate, EB-1053, neridronate, olpadronate, pamidronate, YH 529, tiludronate, or clodronate administered at a dosage ranging from the minimum dose that produces a detectable inhibition of bone resorption up to 0.5 mg P/kg/day. Preferred modes of administration include, but are not limited to, transdermal patch, orally, intravenous injection, subcutaneous injection, and intramuscular injection. The bisphosphonate can be administered as a prophylactic or a therapeutic treatment.
This invention also provides a method of mitigating the symptoms of a disease in a mammal that involves calcification of a soft tissue (an artery, a heart valve, an atherosclerotic plaque, a cancer, a kidney, a prostate, skin, muscle, cartilage, viscera, and heart muscle) The method involves administering to the mammal a low dosage of a bisphosphonate sufficient to inhibit calcification of the soft tissue without inhibiting bone calcification. Such diseases include, but are not limited to atherosclerosis, arterioslerosis, arteriolosclerosis, hypertensive arteriolosclerosis, Monckeberg""s arteriosclerosis, heart valve stenosis, uremia, diabetes, hyperparathyroidism, blood clot formation, cancer growth, cancer metastasis, hypertension, vitamin D toxicity, and arthritis. Preferred bisphosphonates and dosages include, but are not limited to the bisphosphonates and dosages described above. The mammal may be diagnosed as having or at risk for a pathology characterized by calcification of a soft tissue.
In still yet another embodiment, this invention provides methods of mitigating the calcification of an implanted prosthetic device in a mammal. These methods involve administering to the mammal a low dosage of a bisphosphonate sufficient to inhibit calcification of the prosthetic device or soft tissue surrounding said prosthetic device without inhibiting calcification of bone. Such prosthetic devices include, but are not limited to, a heart valve bioprosthesis, and a heart valve mechanical prosthesis. The prosthetic devices can also include, but are not limited to, a surgical implant comprising polyetherurethaneurea, a surgical implant comprising polyetherurethane; a surgical implant comprising silicon, a surgical repair material used for the repair of an aneurisms. Preferred bisphosphonates and dosages include, but are not limited to the bisphosphonates and dosages described above.
The methods of this invention can also be used to mitigate a symptom of atherosclerosis in a mammal. Such methods involve inhibiting osteoclastic bone resorption in said mammal. In preferred embodiment, the inhibiting is by administration of a bisphosphonate to the mammal in a concentration sufficient to inhibit bone resorption without inhibiting bone mineralization. Preferred mammals include, but are not limited to mammals diagnosed as having, or at risk for, atherosclerosis. Preferred bisphosphonates and dosages include, but are not limited to the bisphosphonates and dosages described above. The bisphosphonate is administered as a prophylactic or as a therapeutic treatment.
In another embodiment a symptom or progression of atherosclerosis in a mammal is inhibited by inhibiting the removal of mineral by macrophages at sites of calcification. In a preferred embodiment the inhibiting comprises administering a bisphosphonate to the mammal in a concentration sufficient to inhibit calcium removal by said macrophages. The bisphosphonate is preferably administered at a concentration that does not inhibit macrophages at locations other than sites of calcification. Preferred bisphosphonates and dosages include, but are not limited to the bisphosphonates and dosages described above. The method can be prophylactic and/or therapeutic.
Kits are also provided for the mitigation of a pathology associated with calcification of a soft tissue. Preferred kits include a container containing a bisphosphonate that inhibits calcification of a soft tissue at a dosage that does not substantially inhibit calcification of bone and instructional materials teaching the use of said bisphosphonate for treatment of a pathology associated with calcification of a soft tissue or calcification of a prosthetic device. Preferred bisphosphonates and dosages include, but are not limited to the bisphosphonates and dosages described above.
This invention also provides methods of stabilizing the size and/or the crystal structure of calcium or a calcium salt in an aqueous phase. These methods involve contacting the calcium or calcium salt with fetuin.
The stabilized calcium provides a method of delivering a calcification initiator to a preselected site. Such methods involve providing a fetuin-mineral complex attached to a targeting molecule (e.g., antibody, lectin, nucleic acid etc.) where the targeting molecule specifically binds to the preselected site; and contacting the fetuin mineral complex to the preselected site.
Also provided is a method of distributing mineral nuclei within a matrix. This method involves impregnating the matrix with a fetuin-mineral complex and denaturing the fetuin such that the mineral is released from the fetuin mineral complex.
The fetuin can also be used to stabilize the size or crystal structure of a mineral salt in an aqueous phase. This method involves contacting the mineral salt with a fetuin.
This invention also provides substantially isolated mineral salts (e.g. calcium phosphate) stabilized in a complex with fetuin.
Mammals subject to the methods described herein include, but are not limited to humans, non-human primates, canines, felines, equines, bovines, rodents, porcines, and lagomorphs. Thus, veterinary and human medical applications are contemplated.
In particularly preferred embodiments, the bisphosphonates used in the methods of this invention do not include bisphosphonates for which the dosage that inhibits bone mineralization is comparable to or equal to the dosage that inhibits bone resorption. The bisphosphonates used in the methods of this invention preferably do not include etidronate.
Bisphosphonates, previously and erroneously called diphosphonates in the past, are compounds characterized by two Cxe2x80x94P bonds. If the two bonds are located on the same carbon atom, the compounds are called geminal bisphosphonates and are analogs of pyrophosphate, containing an oxygen instead of a carbon atom (Formula I.). 
In the literature, these compounds are usually called bisphosphonates. This, however, is somewhat misleading, since non-geminal bisphophonates are also bisphosphonates. Thus, as used herein bisphosphonates include, both geminal and non-geminal bisphosphonates.
The Pxe2x80x94Cxe2x80x94P structure allows a great number of possible variations, either by changing the two lateral chains on the carbon or by esterifyng the phosphate groups. A number of bisphosphonates have been investigated in humans with respect to their effects of bone. A number are commercially available for the treatment of bone disease. These include, but are not limited to, alendronate (4-amino-1-hydroxybutylidene)bis-phosphonate), clodronate (dichloromethylene)-bis-phosphonate, EB-1053 (1-hydroxy-3-(1-pyrrolidinyl)-propylidene)bis-phosphonate, etidronate ((1-hydroxyethylylidene)-bisphosphonate), ibandronate (1-hydroxy-3-(methylpentylamino)propylidene)bis-phosphonate), incadronate (([(cycloheptylamino)-methylene]bis-phosphonate), neridronate ((6-amino-1-hydroxyhexylidene)bis-phosphonate), olpadronate ((3-dimethylamino)-1-hydroxypropylidene)bis-phosphonate), palmidronate (3-amino-1-hydroxypropylidene)bis-phosphonate), risedronate (1-hydroxy-2-(3-pyridinyl)-ethylidene)bis-phosphonate), tiludronate ([[(4-chlorophenyl)thio)-methylene]bis-phosphonate), YH 529 ([1-hydroxy-2-imidazo-(1,2-a)pyridin-3-ylethylidene)bis-phosphonate), and zoledronate (1-hydroxy-2-(1H-imidazole-1-y)ethylidene)bis-phosphonate), and the like.
The term xe2x80x9cbone resorptionxe2x80x9d refers to a process by which calcified bone tissue is removed from the bone, e.g. via the activity of osteoclasts. Elevated bone resorption may result in decreased bone mass and/or bone density (e.g. osteoporosis).
The terms xe2x80x9ccalcificationxe2x80x9d refers to the deposition of calcium in a tissue. The calcium can be in a number of forms, e.g. calcium phosphate, hydroxyapatite, carbonate apatite, amorphous calcium phosphate, etc.
The phrase xe2x80x9cinhibition of calcificationxe2x80x9d or xe2x80x9cinhibiting calcificationxe2x80x9d refers to a decrease in the rate and/or degree of calcification of a soft tissue. The inhibition may be complete or partial. Any measurable inhibition is viewed as an inhibition. A preferred inhibition is a statistically significant decrease in the rate and/or degree of calcification (e.g. at the 90% or better, preferably at the 95% or better, more preferably at the 98% or better, and most preferably at the 99% or better confidence level).
The phrase xe2x80x9cwithout inhibiting bone mineralizationxe2x80x9d or xe2x80x9cwithout inhibiting substantial bone mineralizationxe2x80x9d refers to the use of an agent in a dosage that it typically has no substantial effect on bone mineralization. In a preferred embodiment, it typically effects less than a 10%, more preferably less than a 1%, and most preferably less than a 0.1% decrease in the rate of bone mineralization. More preferably it has no statistically significant effect on bone mineralization (e.g. at the 90% or better, preferably at the 95% or better, more preferably at the 98% or better, and most preferably at the 99% or better confidence level). In a most preferred embodiment there is no detectable effect on bone mineralization.
The following abbreviations used are: MGP, matrix Gla protein; BGP, bone Gla protein (osteocalcin); fetuin, xcex12-HS Glycoprotein; and Gla, xcex3-Carboxyglutamic Acid.